4,4-disubstituted cyclohexyl bridged heptamethine cyanine dyes and uses thereof

ABSTRACT

The invention relates to a family of compounds that comprise fluorescent cyanine dyes. The compounds are near infrared absorbing heptamethine cyanine dyes with a 4,4-disubstituted cyclohexyl ring as part of the polymethine chromophore. The compounds are generally hydrophilic and can be chemically linked to biomolecules, such as proteins, nucleic acids, and therapeutic small molecules. The compounds can be used for imaging in a variety of medical, biological and diagnostic applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/215,930, filed Mar. 17, 2014, which claims priority to and thebenefit of U.S. Provisional Patent Application No. 61/798,562, filed onMar. 15, 2013, the contents of each of which are incorporated herein byreference herein in their entireties.

FIELD OF THE INVENTION

The invention provides compositions and methods for new fluorescent dyesthat represent a polymethine bridge comprising a 4,4-disubstitutedcyclohexyl bridged moiety. The new compositions generally containmultiple sulfonic acid or sulfonate groups that render the dye with highhydrophilicity, which can be used in various medical, diagnostic andbiological applications.

BACKGROUND

Optical imaging methods offer a number of advantages over other imagingmethods. Such imaging typically uses light in the red and near-infrared(NIR) range (600-1200 nm) to maximize tissue penetration and minimizeabsorption from natural biological absorbers such as hemoglobin andwater. Optical imaging may provide high sensitivity, does not requireexposure of test subjects or laboratory personnel to ionizing radiation,can allow for simultaneous use of multiple, distinguishable probes(which may be important in molecular imaging), and offers high temporaland spatial resolution, which is important in functional imaging and invivo microscopy, respectively.

In fluorescence imaging, filtered light or a laser with a definedbandwidth is used as a source of excitation light. The excitation lighttravels through body tissue, and when the excitation light encounters areporter molecule (for example, a contrast agent or imaging probe), thelight is absorbed. The reporter molecule then emits light that hasdetectably different properties from the excitation light. The resultingemitted light then can be used to construct an image. Most opticalimaging techniques have relied on the use of organic and inorganicfluorescent dyes as the reporter molecule.

Fluorescent dyes are generally known and used for fluorescence labelingand detection of various biological and non-biological materials byprocedures such as fluorescence microscopy, fluorescence immunoassay,and flow cytometry. A typical method for labeling such materials withfluorescent dyes is to create a fluorescent complex by means of bondingbetween suitable groups on the dye molecule and compatible groups on thematerial to be labeled. In this way, materials such as cells, tissues,amino acids, proteins, antibodies, drugs, hormones, nucleotides, nucleicacids, lipids and polysaccharides and the like may be chemically labeledand detected or quantified, or may be used as fluorescent probes whichcan bind specifically to target materials and detected by fluorescencedetection methods. Brightly fluorescent dyes permit detection orlocalization of the attached materials with great sensitivity.

There is a need for detectable labels for biological and biomedicalresearch. Dyes that work well for quenched probes for use in vivo arenot always effective at in vitro applications. In some cases, thepresence of more than one of these fluorophores on a protein orbiomolecule results in significant quenching which interferes withdetection. There is a need for dyes that will allow for both in vitroand in vivo uses and not over-quench the molecule. Highly soluble,hydrophilic fluorescent dyes would also enable tracking the movement andfunction of labeled cells, proteins, and other biomolecules of interest.A new class of dyes that do not over-quench in vivo or in vitro wouldincrease the tools available for biological research.

Notwithstanding, there is an ongoing need for new dyes that can be usedin various medical, diagnostic and biological applications.

SUMMARY OF THE INVENTION

The invention is based, in part, upon the discovery that it is possibleto produce new fluorescent dyes with a polymethine bridge comprising a4,4-disubstituted cyclohexyl bridged moiety. These dyes can be used in avariety of in vitro and in vivo imaging applications.

In certain embodiments, compounds of the invention can be represented bythe Formula (II)Z¹-(PMB)-Z²  (II), and salts thereof,wherein, Z¹ and Z² each independently can be selected from a substitutedor unsubstituted indolinium or a benzindolinium ring and PMB representsa polymethine bridge comprising a 4,4-disubstituted cyclohexyl bridgedmoiety. In other embodiments, the compounds have an absorption andemission wavelengths in the range from about 500 nm to about 1100 nm,preferably in the range from about 600 nm to about 900 nm. In certainembodiments, the dyes absorb and/or emit light having a wavelength inthe range from about 600 nm to about 850 nm, from about 650 nm to about900 nm, or from about 650 nm to about 850 nm.

In one aspect, the invention provides a family of fluorochrome compoundsthat can be generally represented by Formula I,

In certain embodiments, the invention is a biocompatible fluorescentmolecule represented by the formula III: [BM]_(n)-F_(m), wherein BM is abiomolecule, and F is a fluorophore as described previously. In otherembodiments, the invention is a biocompatible fluorescent biomoleculerepresented by any one of the following structural formulae IVa-IVd,wherein BM is a biomolecule

In another aspect, the invention provides an in vivo optical imagingmethod. The method comprises the steps of (a) administering to asubject, such as an animal or human, a fluorochrome compound of theinvention, (b) allowing the fluorochrome compound to distribute withinthe subject or to contact or interact with a biological target, (c)exposing the subject to electromagnetic radiation, for example, light,of a wavelength absorbable by the fluorochrome compound, and (d)detecting an optical signal emitted by the fluorochrome compound, forexample, with an endoscope, catheter, tomographic system, a planar orreflectance system, hand-held optical imaging system, or intraoperativesystems and microscope. The signal emitted by the compound can be usedto construct an image, for example, a tomographic image, of a region orstructure to be imaged. It is understood that the fluorochrome compoundcan comprise a fluorochrome dye chemically linked to a biomolecule.

The foregoing steps may be repeated at predetermined intervals therebypermitting the evaluation of the emitted signals of the fluorescentcompound in the subject over time. In certain embodiments two or morecompounds whose signal properties are distinguishable can beadministered to the subject and their emission properties can be used toimage two or more features in the subject.

The disclosed methods can be used to detect and/or monitor a disease,for example, bone disease, cancer, cardiovascular disease,dermatological disease, environmental disease, immunologic disease,infectious disease, inflammation, inherited disease, metabolic disease,neurodegenerative disease, ophthalmic disease, and respiratory disease.

In certain embodiments, cells are labeled with a fluorochrome compounddescribed herein and the resulting labeled cells administered to thesubject. The signal emitted by the fluorochrome compound can be used tomonitor transport and localization of the cells or to evaluate theefficacy of a cell therapy.

In another aspect, the invention provides an in vitro optical imagingmethod. The method comprises the steps of (a) contacting a sample, forexample, a biological sample, with the fluorochrome compound of theinvention, (b) allowing the fluorochrome compound to become activated byor to bind to a biological target; (c) optionally, removing unboundfluorochrome compound; (d) exposing the sample to electromagneticradiation, for example, light, of a wavelength absorbable by thefluorochrome compound; and (e) detecting signal emitted from thefluorochrome compound thereby to determine whether the fluorochromecompound has been activated by or bound to the biological target.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict the fluorescence and absorbance spectra forBovine Serum Albumin (BSA)-conjugated dyes of the present invention.FIG. 1A is a graph of fluorescence emitted by the BSA-conjugate ofCompound 1b. FIG. 1B depicts fluorescence absorbance of BSA-conjugate ofCompound 1b conjugated to BSA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a family of fluorochrome compounds (dyes)that absorb and/or emit light having a wavelength in the range fromabout 500 nm to about 1100 nm, more preferably in the range from about600 nm to about 900 nm. In certain embodiments, the dyes absorb and/oremit light having a wavelength in the range from about 600 nm to about850 nm, from about 650 nm to about 900 nm, or from about 650 nm to about850 nm. The fluorochrome compounds are particularly useful in a varietyof in vitro and in vivo imaging applications.

In certain embodiments, the fluorochrome compounds of the invention canbe represented by the formula Z¹—PMB-Z², and salts thereof, wherein Z¹and Z² each independently represent the same or different polycyclicgroups containing a heterocyclic moiety, and PMB represents apolymethine bridge comprising a 4,4-disubstituted cyclohexyl bridgedmoiety. The fluorochrome compounds will be discussed in more detailherein below. However, before further description of the presentinvention, certain terms employed in the specification, examples andappended claims are collected together in the following section.

I. Definitions

The definitions listed herein should be read in light of the remainderof the disclosure and understood as by a person of skill in the art.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art to which this invention belongs.

“Chemically linked” means connected by an attractive force between atomsstrong enough to allow the combined aggregate to function as a unit.This includes, but is not limited to, chemical bonds such as covalentbonds, non-covalent bonds such as ionic bonds, metallic bonds, andbridge bonds, hydrophobic interactions, hydrogen bonds, and van derWaals interactions. This also includes crosslinking or caging.

The term “alkyl” is art-recognized, and includes saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups. In certain embodiments,a straight chain or branched chain alkyl has about 30 or fewer carbonatoms in its backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ forbranched chain), and alternatively, about 20 or fewer. Likewise,cycloalkyls have from about 3 to about 10 carbon atoms in their ringstructure, and alternatively about 5, 6 or 7 carbons in the ringstructure. The term “alkyl” also includes halosubstituted alkyls.

Moreover, the term “alkyl” includes “substituted alkyls”, which refersto alkyl moieties having substituents replacing a hydrogen on one ormore carbons of the hydrocarbon backbone. Such substituents may include,for example, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphonate, a phosphinate, an amino, an amido, an amidine, an imine, acyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, asulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, anaralkyl, or an aromatic or heteroaromatic moiety. It will be understoodby those skilled in the art that the moieties substituted on thehydrocarbon chain may themselves be substituted, if appropriate. Forinstance, the substituents of a substituted alkyl may includesubstituted and unsubstituted forms of amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CN and the like. Exemplary substitutedalkyls are described below. Cycloalkyls may be further substituted withalkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substitutedalkyls, —CN, and the like. In certain embodiments, the alkyl isunsubstituted. In certain embodiments, the alkyl is a straight orbranched chain alkyl group that is unsubstituted.

The term “haloalkyl” refers to an alkyl group as defined above exceptthat one or more hydrogen atoms have been replaced with a halogen.

The term “alkylene” refers to a diradical of a straight or branchedchain alkyl group that is unsubstituted.

The terms “aralkyl” and “alkylaryl” are art-recognized and refer to analkyl group substituted with an aryl group (e.g., an aromatic orheteroaromatic group).

The terms “alkenyl” and “alkynyl” are art-recognized and refer tounsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but that contain at leastone double or triple bond, respectively.

The term “heteroatom” is art-recognized and refers to an atom of anyelement other than carbon or hydrogen. Illustrative heteroatoms includeboron, nitrogen, oxygen, phosphorus, sulfur and selenium.

The term “aryl” is art-recognized and refers to 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “heteroaryl” or“heteroaromatics.” The aromatic ring may be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

The terms “heterocyclyl,” “heterocyclic group” or “heterocyclic moiety”are art-recognized and refer to 3- to about 10-membered ring structures,alternatively 3- to about 7-membered rings, whose ring structuresinclude one to four heteroatoms. Heterocycles may also be polycycles.Heterocyclyl groups include, for example, thiophene, thianthrene, furan,pyran, isobenzofuran, chromene, xanthene, phenoxanthene, pyrrole,imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, and thelike. The heterocyclic ring may be substituted at one or more positionswith such substituents as described above, as for example, halogen,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The terms “polycyclyl,” “polycyclic group” or “polycyclo moiety” areart-recognized and refer to two or more rings (e.g., cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which twoor more carbons are common to two adjoining rings, e.g., the rings are“fused rings.” Rings that are joined through non-adjacent atoms aretermed “bridged” rings. Each of the rings of the polycycle may besubstituted with such substituents as described above, as for example,halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino,nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl,carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The term “nitro” is art-recognized and refers to —NO₂; the term“halogen” is art-recognized and refers to —F, —Cl, —Br or —I; the term“sulfhydryl” is art-recognized and refers to —SH; the term “hydroxyl”means —OH; and the term “sulfonyl” is art-recognized and refers to —SO₂⁻. “Halide” designates the corresponding anion of the halogens, and“pseudohalide” has the definition set forth in “Advanced InorganicChemistry” by Cotton and Wilkinson.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that may berepresented by the general formulas:

wherein R₅₀, R₅₁, R₅₂ and R₅₃ each independently represent a hydrogen,an alkyl, an alkenyl, —(CH₂)_(m)—R₆₁, or R₅₀ and R₅₁, taken togetherwith the N atom to which they are attached complete a heterocycle havingfrom 4 to 8 atoms in the ring structure; R₆₁ represents an aryl, acycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zeroor an integer in the range of 1 to 8. In certain embodiments, only oneof R₅₀ or R₅₁ may be a carbonyl, e.g., R₅₀, R₅₁ and the nitrogentogether do not form an imide. In other embodiments, R₅₀ and R₅₁ (andoptionally R₅₂) each independently represent a hydrogen, an alkyl, analkenyl, or —(CH₂)_(m)—R₆₁. Thus, the term “alkylamine” includes anamine group, as defined above, having a substituted or unsubstitutedalkyl attached thereto, i.e., at least one of R₅₀ and R₅₁ is an alkylgroup.

The term “acylamino” is art-recognized and refers to a moiety that maybe represented by the general formula:

wherein R₅₀ is as defined above, and R₅₄ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₆₁, where m and R₆₁ are as definedabove.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that may be represented by the general formula:

wherein R₅₀ and R₅₁ are as defined above. Certain embodiments of theamide in the present invention will not include imides which may beunstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In certain embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₆₁, wherein m and R₆₁ are defined above.Representative alkylthio groups include methylthio, ethylthio, and thelike.

The term “carbonyl” is art recognized and includes such moieties as maybe represented by the general formulas:

wherein X₅₀ is a bond or represents an oxygen or a sulfur, and R₅₅ andR₅₆ represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₆₁ or apharmaceutically acceptable salt, R₅₆ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₆₁, where m and R₆₁ are defined above. WhereX₅₀ is an oxygen and R₅₅ or R₅₆ is not hydrogen, the formula representsan “ester.” Where X₅₀ is an oxygen, and R₅₅ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₅₅ is a hydrogen, the formula represents a “carboxylic acid.” Where X₅₀is an oxygen, and R₅₆ is hydrogen, the formula represents a “formate.”In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X₅₀ is asulfur and R₅₅ or R₅₆ is not hydrogen, the formula represents a“thiolester.” Where X₅₀ is a sulfur and R₅₅ is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X₅₀ is a sulfur and R₅₆ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X₅₀ is a bond, and R₅₅ is not hydrogen, the above formularepresents a “ketone” group. Where X₅₀ is a bond, and R₅₅ is hydrogen,the above formula represents an “aldehyde” group.

The terms “alkoxyl” or “alkoxy” are art-recognized and refer to an alkylgroup, as defined above, having an oxygen attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as may berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl,—O—(CH₂)_(m)—R₆₁, where m and R₆₁ are described above.

The term “sulfonate” is art recognized and refers to a moiety that maybe represented by the general formula:

in which R₅₇ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The term “sulfate” is art recognized and includes a moiety that may berepresented by the general formula:

in which R₅₇ is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that maybe represented by the general formula:

in which R₅₀ and R₅₆ are as defined above.

The term “sulfamoyl” is art-recognized and refers to a moiety that maybe represented by the general formula:

in which R₅₀ and R₅₁ are as defined above.

The term “sulfonyl” is art-recognized and refers to a moiety that may berepresented by the general formula:

in which R₅₈ is one of the following: hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclyl, aryl or heteroaryl.

The term “sulfoxido” is art-recognized and refers to a moiety that maybe represented by the general formula:

in which R₅₈ is defined above.

The term “phosphoryl” is art-recognized and may in general berepresented by the formula:

wherein Q₅₀ represents S or O, and R₅₉ represents hydrogen, a loweralkyl or an aryl. When used to substitute, e.g., an alkyl, thephosphoryl group of the phosphorylalkyl may be represented by thegeneral formulas:

wherein Q₅₀ and R₅₉, each independently, are defined above, and Q₅₁represents 0, S or N. When Q₅₀ is S, the phosphoryl moiety is a“phosphorothioate”.

The term “phosphoramidite” is art-recognized and may be represented inthe general formulas:

wherein Q₅₁, R₅₀, R₅₁ and R₅₉ are as defined above.

The term “phosphonamidite” is art-recognized and may be represented inthe general formulas:

wherein Q₅₁, R₅₀, R₅₁ and R₅₉ are as defined above, and R₆₀ represents alower alkyl or an aryl.

Analogous substitutions may be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

The definition of each expression, e.g., alkyl, m, n, and the like, whenit occurs more than once in any structure, is intended to be independentof its definition elsewhere in the same structure.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction.

The term “substituted” is also contemplated to include all permissiblesubstituents of organic compounds. Exemplary substituents include, forexample, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, and the like. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentsmay be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

The term “polymethine bridge” refers to a conjugated double bondmethylene chain comprising an odd number of carbons. Such a bridge caninclude a ring structure as part of the conjugated double bond methylenechain.

The term “physiologically acceptable carrier” refers to a carrier inwhich one or more of the compounds of the invention are dispersed,dissolved, suspended, admixed and physiologically tolerable, i.e., canbe administered to, in, or on the subject's body without unduediscomfort, or irritation, or toxicity.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where processes are described as having,including, or comprising specific process steps, the processes alsoconsist essentially of, or consist of, the recited processing steps.Further, it should be understood that the order of steps or order forperforming certain actions are immaterial so long as the inventionremains operable. Moreover, two or more steps or actions may beconducted simultaneously.

II. Fluorochrome Compounds of the Invention

One aspect of the invention provides a fluorescent compound representedby Formula I-A:

or a salt thereof, wherein:

X₁ and X₂ are each independently O, S, Se, or C(C₁₋₄ alkyl)₂;

W₁ and W₂ are a benzo, naphtha, or pyridyl ring;

R₁ and R₂ are independently hydrogen or —C₁-C₁₀ alkyl optionallysubstituted with one or two substituents independently selected from thegroup consisting of halogen, —SO₃H, —SO₃ ⁻, —COOH, —CO₂ ⁻, and —OH;

R₅, R₆, R₇ and R₈ are each independently H or —C₁-C₂₂ alkylene-X₃;

R₃, R₄, R₁₃ and R₁₄ are each independently H, —C₁-C₂₂ alkylene-X₃,—SO₃H, —SO₃ ⁻, —SO₂N(R₁₂)-alkylene-X₃, halogen, or —NO₂;

X₃ represents independently for each occurrence H, halogen, —CH₃, —SO₃H,—SO₃ ⁻, —COOH, —CO₂ ⁻, —NCS, —NCO, N-hydroxysuccinimidyl ester,N-hydroxysulfosuccinimidyl ester, —OH, —SH, maleimide, phthalimide,—NHCO—(CH₂)_(m)-(halogen), —CONHNH₂, —CN, —NH₂, —NO₂, —CON(H)R₁₂,alkynyl, —N₃, a polyethyl glycol, optionally substituted aryl, oroptionally substituted heterocyclyl;

R₉ and R₁₀ are hydrogen, halogen, or alkyl, or R₁ and R₉ or R₂ and R₁₀are taken together with their interconnecting atoms to form a 5-, 6- or7-membered ring;

R₁₂ represents independently for each occurrence hydrogen or alkyl;

m represents independently for each occurrence 0, 1, 2, 3, or 4; and

n represents independently for each occurrence 1-10.

In certain embodiments, X₁ and X₂ are C(CH₃)₂. In certain embodiments,W₁ and W₂ are a benzo ring. In certain embodiments, W₁ and W₂ are anaptha ring. In certain embodiments, R₁ and R₂ are independently —C₁-C₁₀alkyl optionally substituted with —SO₃H or —SO₃ ⁻. In certainembodiments, R₁ and R₂ are independently C₁-C₆ alkyl. In certainembodiments, R₃, R₄, R₁₃ and R₁₄ are each independently H, —SO₃H, or—SO₃. In certain embodiments, R₇ is hydrogen. In certain embodiments, R₉and R₁₀ are hydrogen.

In certain embodiments, R₅ and R₆ are each independently —C₁-C₂₂alkylene-X₃. In certain embodiments, R₅ and R₆ are each independently—C₂-C₈ alkylene-X₃. In certain embodiments, R₅ and R₆ are eachindependently —C₂-C₈ alkylene substituted by —SO₃H, —SO₃ ⁻, or —COOH. Incertain embodiments, R₇ and R₈ are hydrogen.

Another aspect of the invention provides a fluorescent compoundrepresented by Formula I-B:

or a salt thereof, wherein:

X₁ and X₂ are each independently O, S, Se, or C(C₁₋₄ alkyl)₂;

W₁ and W₂ are a benzo, naphtha, or pyridyl ring;

R₁ and R₂ are independently hydrogen or —C₁-C₁₀ alkyl optionallysubstituted with one or two substituents independently selected from thegroup consisting of halogen, —SO₃H, —SO₃ ⁻, —COOH, —CO₂ ⁻, and —OH;

R₅ and R₇ are each independently hydrogen or —C₁-C₂₂ alkylene-X₃;

R₃, R₄, R₁₃ and R₁₄ are each independently hydrogen, —C₁-C₂₂alkylene-X₃, —SO₃H, —SO₃ ⁻, —SO₂N(R₁₂)-alkylene-X₃, halogen, or —NO₂;

X₃ represents independently for each occurrence H, halogen, —CH₃, —SO₃H,—SO₃ ⁻, —COOH, —CO₂ ⁻, —NCS, —NCO, N-hydroxysuccinimidyl ester,N-hydroxysulfosuccinimidyl ester, —OH, —SH, maleimide, phthalimide,—NHCO—(CH₂)_(m)-(halogen), —CONHNH₂, —CN, —NH₂, —NO₂, —CON(H)R₁₂,alkynyl, —N₃, a polyethyl glycol, optionally substituted aryl, oroptionally substituted heterocyclyl;

R₉ and R₁₀ are H, halogen, or alkyl, or R₁ and R₉ or R₂ and R₁₀ aretaken together with their interconnecting atoms to form a 5-, 6- or7-membered ring;

R₁₁ is —COOH, —CN, halogen, —NO₂, —C(O)-haloalkyl, haloalkyl, —COOR₁₅,—CON(H)R₁₅, or —CO(CH₂)_(n)R₁₅;

R₁₂ represents independently for each occurrence hydrogen or alkyl;

R₁₅ is H, —COOH, —SO₃H, —NH₂, —SH, alkyl, or aryl optionally substitutedwith X₃ and/or a polyethylene glycol;

m represents independently for each occurrence 0, 1, 2, 3, or 4; and

n represents independently for each occurrence 1-10.

In certain embodiments, X₁ and X₂ are C(CH₃)₂. In certain embodiments,W₁ and W₂ are a benzo ring. In certain embodiments, W₁ and W₂ are anaptha ring. In certain embodiments, R₁ and R₂ are independently —C₁-C₁₀alkyl optionally substituted with —SO₃H or —SO₃ ⁻. In certainembodiments, R₁ and R₂ are independently C₁-C₆ alkyl. In certainembodiments, R₃, R₄, R₁₃ and R₁₄ are each independently H, —SO₃H, or—SO₃. In certain embodiments, R₇ is hydrogen. In certain embodiments, R₉and R₁₀ are hydrogen.

In certain embodiments, R₅ is —C₁-C₂₂ alkylene-X₃, and R₇ is hydrogen.In certain embodiments, R₅ is —C₂-C₈ alkylene-X₃, and R₇ is hydrogen. Incertain embodiments, R₅ is —C₂-C₈ alkylene substituted by —SO₃H, —SO₃ ⁻,or —COOH, and R₇ is hydrogen.

Another aspect of the invention provides a fluorescent compoundrepresented by Formula I-C:

or a salt thereof, wherein:

X₁ and X₂ are each independently O, S, Se, or C(C₁₋₄ alkyl)₂;

R₁ and R₂ are independently hydrogen or —C₁-C₁₀ alkyl optionallysubstituted with one or two substituents independently selected from thegroup consisting of halogen, —SO₃H, —SO₃ ⁻, —COOH, —CO₂ ⁻, and —OH;

R₃, R₄, R₁₃ and R₁₄ are each independently hydrogen, —C₁-C₂₂alkylene-X₃, —SO₃H, —SO₃ ⁻, —SO₂N(R₁₂)-alkylene-X₃, halogen, or —NO₂;

X₃ represents independently for each occurrence H, halogen, —CH₃, —SO₃H,—SO₃ ⁻, —COOH, —CO₂ ⁻, —NCS, —NCO, N-hydroxysuccinimidyl ester,N-hydroxysulfosuccinimidyl ester, —OH, —SH, maleimide, phthalimide,—NHCO—(CH₂)_(m)-(halogen), —CONHNH₂, —CN, —NH₂, —NO₂, —CON(H)R₁₂,alkynyl, —N₃, a polyethyl glycol, optionally substituted aryl, oroptionally substituted heterocyclyl;

X₄ represents independently for each occurrence hydrogen, halogen,—SO₃H, —SO₃ ⁻, —COOH, or —CO₂ ⁻;

R₉ and R₁₀ are H, halogen, or alkyl, or R₁ and R₉ or R₂ and R₁₀ aretaken together with their interconnecting atoms form a 5-, 6- or7-membered ring;

R₁₂ represents independently for each occurrence hydrogen or alkyl;

m represents independently for each occurrence 0, 1, 2, 3, or 4; and

n represents independently for each occurrence 1-10.

In certain embodiments, X₁ and X₂ are C(CH₃)₂. In certain embodiments,W₁ and W₂ are a benzo ring. In certain embodiments, W₁ and W₂ are anaptha ring. In certain embodiments, R₁ and R₂ are independently —C₁-C₁₀alkyl optionally substituted with —SO₃H or —SO₃ ⁻. In certainembodiments, R₁ and R₂ are independently C₁-C₆ alkyl. In certainembodiments, R₃, R₄, R₁₃ and R₁₄ are each independently H, —SO₃H, or—SO₃. In certain embodiments, R₇ is hydrogen. In certain embodiments, R₉and R₁₀ are hydrogen.

Another aspect of the invention provides a fluorescent compoundrepresented by Formula I-D:

or a salt thereof, wherein:

X₁ and X₂ are each independently O, S, Se, or C(C₁₋₄ alkyl)₂;

W₁ and W₂ are a benzo, naphtha, or pyridyl ring;

R₁ and R₂ are independently hydrogen or —C₁-C₁₀ alkyl optionallysubstituted with one or two substituents independently selected from thegroup consisting of halogen, —SO₃H, —COOH, —CO₂ ⁻, and —OH;

R₃, R₄, R₁₃ and R₁₄ are each independently H, —C₁-C₂₂ alkylene-X₃,—SO₃H, —SO₂N(R₁₂)-alkylene-X₃, halogen, or —NO₂;

X₃ represents independently for each occurrence H, halogen, —CH₃, —SO₃H,—SO₃ ⁻, —COOH, —CO₂ ⁻, —NCS, —NCO, N-hydroxysuccinimidyl ester,N-hydroxysulfosuccinimidyl ester, —OH, —SH, maleimide, phthalimide,—NHCO—(CH₂)_(m)-(halogen), —CONHNH₂, —CN, —NH₂, —NO₂, —CON(H)R₁₃,alkynyl, —N₃, a polyethyl glycol, optionally substituted aryl, oroptionally substituted heterocyclyl;

R₉ and R₁₀ are hydrogen, halogen, or alkyl, or R₁ and R₉ or R₂ and R₁₀are taken together with their interconnecting atoms to form a 5-, 6- or7-membered ring;

R₁₁ and R₁₂ are each independently alkyl, haloalkyl, aryl, aralkyl,cyano, halogen, nitro, —COOH, —C(O)-haloalkyl, —C(O)-aryl, —C(O)OR₁₅,—CON(H)R₁₅, —(CH₂)_(n)C(O)OR₁₅, —(CH₂)_(n)CONHR₁₅, —CO(CH₂)_(n)R₁₅,—(CH₂)_(n)SO₃H, or —(CH₂)_(n)SO₃ ⁻,

R₁₃ represents independently for each occurrence hydrogen or alkyl;

R₁₅ represents independently for each occurrence H, —COOH, —SO₃H, —NH₂,—SH, alkyl, a polyethylene glycol, or aryl which may be optionallysubstituted with X₃ and/or a polyethylene glycol;

m represents independently for each occurrence 0, 1, 2, 3, or 4; and

n represents independently for each occurrence 1-10.

In certain embodiments, X₁ and X₂ are C(CH₃)₂. In certain embodiments,W₁ and W₂ are a benzo ring. In certain embodiments, W₁ and W₂ are anaptha ring. In certain embodiments, R₁ and R₂ are independently —C₁-C₁₀alkyl optionally substituted with —SO₃H or —SO₃ ⁻. In certainembodiments, R₁ and R₂ are independently C₁-C₆ alkyl. In certainembodiments, R₃, R₄, R₁₃ and R₁₄ are each independently H, —SO₃H or—SO₃. In certain embodiments, R₇ is hydrogen. In certain embodiments, R₉and R₁₀ are hydrogen.

Another aspect of the invention provides a fluorescent compoundrepresented by Formula II:

or a salt thereof, wherein:

R₁ and R₂ are independently hydrogen or —C₁-C₁₀ alkyl optionallysubstituted with one or two substituents independently selected from thegroup consisting of halogen, —SO₃H, —SO₃ ⁻, —COOH, —CO₂ ⁻, and —OH;

R₅, R₆, R₇ and R₈ are each independently H or —C₁-C₂₂ alkylene-X₃;

R₃, R₄, R₁₃ and R₁₄ are each independently H, —C₁-C₂₂ alkylene-X₃,—SO₃H, —SO₃ ⁻, —SO₂N(R₁₂)-alkylene-X₃, halogen, or —NO₂;

X₃ represents independently for each occurrence H, halogen, —CH₃, —SO₃H,—SO₃ ⁻, —COOH, —CO₂ ⁻, —NCS, —NCO, N-hydroxysuccinimidyl ester,N-hydroxysulfosuccinimidyl ester, —OH, —SH, maleimide, phthalimide,—NHCO—(CH₂)_(m)-(halogen), —CONHNH₂, —CN, —NH₂, —NO₂, —CON(H)R₁₂,alkynyl, —N₃, a polyethyl glycol, optionally substituted aryl, oroptionally substituted heterocyclyl;

R₉ and R₁₀ are hydrogen, halogen, or alkyl, or R₁ and R₉ or R₂ and R₁₀are taken together with their interconnecting atoms to form a 5-, 6- or7-membered ring;

R₁₂ represents independently for each occurrence hydrogen or alkyl;

m represents independently for each occurrence 0, 1, 2, 3, or 4; and

n represents independently for each occurrence 1-10.

In certain embodiments, R₁ and R₂ are independently —C₁-C₁₀ alkyloptionally substituted with —SO₃H or —SO₃ ⁻. In certain embodiments, R₁and R₂ are independently —C₂-C₆ alkyl optionally substituted with —SO₃Hor —SO₃. In certain embodiments, R₁ and R₂ are independently C₁-C₆alkyl. In certain embodiments, R₅ and R₆ are each independently —C₁-C₂₂alkylene-X₃. In certain embodiments, R₅ and R₆ are each independently—C₂-C₈ alkylene-X₃. In certain embodiments, R₅ and R₆ are eachindependently —C₂-C₈ alkylene substituted by —SO₃H, —SO₃ ⁻, or —COOH. Incertain embodiments, R₇ and R₈ are hydrogen. In certain embodiments, R₉and R₁₀ are hydrogen.

Another aspect of the invention provides compounds represented by theFormula (II)Z¹-(PMB)-Z²  (II), and salts thereof.

Z¹ and Z² each independently represent a polycyclic group comprising aheterocyclic moiety. For example, Z¹ and Z² each independently can beselected from a substituted or unsubstituted indolinium or abenzindolinium ring. PMB represents a polymethine bridge comprising a4,4-disubstituted cyclohexyl bridged moiety. The compounds have anabsorption and emission wavelengths in the range from about 500 nm toabout 1100 nm, preferably in the range from about 600 nm to about 900nm. In certain embodiments, the dyes absorb and/or emit light having awavelength in the range from about 600 nm to about 850 nm, from about650 nm to about 900 nm, or from about 650 nm to about 850 nm.

Z¹, Z², and/or PMB optionally can include a linker moiety capable offorming a covalent bond, and/or chemical linkage to a biomolecule. Sucha linker moiety can include a reactive group that is capable ofchemically reacting with a functional group on a different compound toform a covalent linkage, or a functional group that is capable ofchemically reacting with a reactive group on different compound to forma covalent linkage. Such a reactive group can include, for example, anelectrophile or nucleophile that can form a covalent linkage viaexposure to a corresponding functional group that is a nucleophile orelectrophile, respectively. Alternatively, the reactive group is aphotoactivatable group, and becomes chemically reactive only afterillumination with light of an appropriate wavelength. A reaction betweenthe compound of the invention and the biomolecule to be linked canresult in one or more atoms of a reactive group incorporated into a newlinkage attaching a compound of the invention to the conjugatedsubstance.

Biomolecules contemplated herein include, but are not limited to,proteins (for example, enzymes, hormones, antibodies and antigen bindingfragments thereof, and single chain antibodies), peptides, amino acids,glycoproteins, ligands for cell receptors, polysaccharides,carbohydrates, nucleic acids (for example, DNA and RNA), nucleosides,nucleotides, aptamers, peptidyl nucleic acids, cell receptors, enzymesubstrates, enzyme cofactors, biotin, hormones, neurotransmitters,growth factors, cytokines, lymphokines, lectins, selectins, lipids,lipid assemblies (for example, micelles or vesicles), and toxins. Otherbiomolecules can be used, such as those involved in targeting anddelivery such as folate-mediated targeting (Leamon & Low, Drug DiscoveryToday, 6:44-51, 2001), transferrin, vitamins, carbohydrates and ligandsthat target internalizing receptors, including, but not limited to,asialoglycoprotein receptor, somatostatin, nerve growth factor,oxytocin, bombesin, calcitonin, arginine vasopressin, angiotensin II,atrial natriuretic peptide, insulin, glucagons, prolactin, gonadotropin,various opioids and urokinase-type plasminogen activator. Alsocontemplated are membrane, transmembrane, and nuclear translocationsignal sequences, which can be derived from a number of sourcesincluding, without limitation, viruses and bacteria. Biomolecules canalso include organic molecules, polymers, dendrimers, cells (forexample, mammalian cells, non mammalian cells, plant cells, insectcells, embryonic cells), bacteria, bacteriophage, viruses, organisms,particles, microparticles, or nanoparticles. Biomolecules can alsoinclude therapeutic drug molecules including but not limited tophototherapy or radiotherapy molecules.

The fluorochrome compounds of the present invention can be used tocreate one or more of the following types of imaging agents or probes: amolecular probe, an activatable probe, an enzyme-activatable probe, aquantum dot-based imaging probe, a nanoparticle-based imaging probe, aprobe targeted to a biomolecule, a wavelength shifting beacon, amulticolor probe, a probe with high binding affinity to a target, anon-specific imaging probe, cell based probe, a dual modality agent, anoptical/CT dual modality agent (e.g., an optical agent physically orchemically bound to a CT agent), an optical/MR dual modality agent(e.g., an optical agent physically or chemically bound to an MR agent),an optical/nuclear dual modality agent (e.g., an optical agentphysically or chemically bound or with a radioactive atom) and/or anycombination thereof.

Compounds of the invention that include a chemically linked biomoleculemay have enhanced fluorescence as compared to the compound that is notchemically linked to a biomolecule. In certain embodiments, thefluorescence is enhanced by about 10%, about 25% or about 50% whencompared with the unlinked compound. Biomolecules chemically linked tothe compounds of the invention may alter or enhance accumulation,biodistribution, elimination, targeting, binding, and/or recognition ofthe molecules in vivo and/or in vitro.

One or more biomolecules may be chemically linked to Z¹, PMB, and/or Z²via multivalent linkages or linkers containing several reactivefunctional groups to form a biocompatible fluorescent molecule of thestructure (Z¹-(PMB)-Z²)-((L)_(v)(BM)_(r))_(t), wherein L is a linker orspacer or multivalent spacer or linker, BM is a biomolecule, Z¹, Z² andPMB are as previously defined, and t=1-6, v=1-500 and r=1-500. (L)_(v),when v is greater than 1, represents copies of the same linker or acombination of different linkers.

Examples of appropriate linker moieties for compounds of the presentinvention have been previously described in the literature (see, U.S.Patent Appl. 2002/0064794 (2002); U.S. Pat. Nos. 6,086,737; 6,048,982;6,747,159; and 6,448,008).

It is understood that more than one fluorochrome compound of the presentinvention can be chemically linked to a single biomolecule. An exampleof such a structure can be represented as: [Z¹-(PMB)-Z²]_(u)-BM, whereinu=1-500 and Z¹, Z², PMB and BM are as defined above.

Salts of the disclosed compounds are also contemplated, and include bothbase and acid addition salts. The compounds of the present invention canhave one or more sufficiently acidic proton that can react with asuitable organic or inorganic base to form a base addition salt. Baseaddition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like, and organic bases such as alkoxides, alkylamides, alkyl and aryl amines, and the like. Such bases useful inpreparing the salts of this invention thus include sodium hydroxide,potassium hydroxide, ammonium hydroxide, potassium carbonate, and thelike.

The compounds of the present invention having a sufficiently basicgroup, such as an amine can react with an organic or inorganic acid toform an acid addition salt. Acids commonly employed to form acidaddition salts from compounds with basic groups are inorganic acids suchas hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid,phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of such salts includethe sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like.

For example, compounds of Formula I can be represented by formulae Ia,Ib and Ic

or a salt thereof, wherein:Wherein X₁ and X₂ are independently chosen from O, S, Se, C(CH₂R₃CH₂R₄);R₁, R₂, R₅, R₆, R₇ and R₈ are each independently chosen from: H,(CH₂)_(n)X₃, wherein n=1-20; R₃, R₄, R₁₃ and R₁₄ are each independentlychosen from: H, (CH₂)_(n)X₃, wherein n=0-20; X₃ is independently chosenfrom: H, halogen, CH₃, SO₃H, SO₃—, COOH, NCS (isothiocyanate), NCO(iscocyanate), N-hydroxy succinimidyl (NHS) ester,N-hydroxysulfosuccinimidyl (NHSS) ester, hydroxy (OH), thiol (SH),maleimide, phthalimide, iodoacetamide, CN, NH₂, CONHR, alkyne, azide(N₃), SO₂NX₃R₇, aryl that is optionally further substituted with X₃; R₉and R₁₀ are H or halogen or alkyl group; R₁ and R₉ or R₂ and R₁₀optionally taken together form a 5 or 6 or 7 membered ring; W₁ and W₂are the atoms necessary to form aryl rings including benzo or naphtho orpyridyl; R₁₁ is independently chosen from: COOH, CN, F, NO₂, COCF₃, CF₃,COOR, CONHR, CO(CH₂)_(n)R, wherein R is H or COOH or SO₃H, or NH₂ or SHor alkyl or aryl which is optionally further substituted with X₃, orpolyethylene glycol (PEG) units

In certain embodiments, X₃ is selected from the group consisting of—NH₂, —OH, —SH, —SO₃H, carboxyl, —COCl, —(CO)O(CO)R₁₆, —CONHNH₂,substituted and unsubstituted N-hydroxysuccinimido esters, substitutedand unsubstituted N-hydroxysulfosuccinimido esters, nitro- orfluoro-phenol esters, azide, —NCS, —CHO, azide, —COCH₂I,phosphoramidite, phthalamido, and maleimide, wherein R₁₆ is selectedfrom the group consisting of H, alkyl and aryl.

In other embodiments, X₁ and X₂ are —C(CH₃)₂.

It is understood that W₁ and W₂ may be the same or different. Forexample, W₁ and W₂ can be selected from the group consisting of:

Incorporation of one or more non-hydrogen substituents on the fusedrings can be used to tune the absorption and emission spectrum of theresulting dye.

In certain embodiments, the compounds is one of the following or a saltthereof:

R₁₁ and R₁₂ are independently: COOH, CONHR, CN, O═C-Phenyl, COCH₂R whereR═H or

(CH₂)_(n)COOR′ or (CH₂)nCH₃ or (CH₂)_(n)SO₃H or (CH₂)_(n)SO₃ ⁻, whereR′=alkyl or aryl

When a compound of the invention is depicted herein by structureindicating the positions of the double bonds in the rings andpolymethine bridge, it is to be understood that the structure alsoencompasses any resonance structures as shown, for example, in thefigure below:

wherein, in each of the foregoing structures, R₁, R₂, R₃, R₄, R₅, R₆,R₇, R₈, R₉, R₁₀, R₁₃, R₁₄, W₁, W₂, X₁, X₂, and X₃ are as defined herein.

Generally, the compounds disclosed herein can be synthesized as follows.First, a quaternized heterocycle, Z¹, is prepared. Then, theheterocyclic base is reacted with a polymethine bridge (PMB) that is anelectrophilic reagent, such as PhNH-PMB-CH═NHPh.HCl, or RO-PMB—CH(OR)₂,where PMB consists of a conjugated double bond chain (CH═CH)_(n)— thatincludes a 4,4-disubstituted cyclohexyl bridged moiety as part of suchchain, and where Ph is a phenyl ring and R a methyl or ethyl group, toobtain hemicyanines such as Z¹—PMB-CH═NHPh or Z¹—PMB-CH═NAcPh (where Acis the acetyl radical) or Z¹—(CH═CH)_(n)—OR. These intermediates thenare reacted with a different quaternary heterocycle, Z². Thefunctionalized side arm is attached either to the first (Z¹) or to thesecond (Z²) quaternized heterocycle. The final result is a non-symmetricpolymethine labeling reagent, Z¹-PMB-Z². Examples of hemicyanineintermediates are described in F. M. Hamer, “Some UnsymmetricalPentamethincyanine Dyes and their Tetramethin Intermediates”, J. Chem.Soc., 32 (1949) and R. B. Mujumdar, L. A. Ernst, Swati R. Mujumdar, C.J. Lewis, and A. S. Waggoner, “Cyanine Dye Labelling Reagents:Sulfoindocyanine Succinimidyl Esters”, Bioconjugate Chemistry, 4, 105,(1993).

In another aspect, the invention provides compounds of generalstructural formula V

wherein R₁₁ and R₁₂ are independently: COOH, CONHR, CF₃, halogen, CN,O═C-Phenyl, COCH₂R where R═H or

(CH₂)_(n)COOR′ or (CH₂)nCH₃ or (CH₂)_(n)SO₃H or (CH₂)_(n)SO₃ ⁻, whereR′=alkyl or aryl; Ph is phenyl group, which is optionally substitutedwith one of: F, Cl, Br, I, OMe, NMe₂, NO₂, CN, CF₃, alkyl.

The certain other embodiments, following structure represented byformula 45a and 45b are contemplated, wherein R′ is alkyl or aryl

In certain embodiments, the compounds of the invention can be chemicallylinked to a biological molecule or biomolecule (BM) as represented byformula III—[BM]_(n)-F_(m), wherein BM is a biomolecule, F is afluorophore represented by formulae 1a, 1b or 1c (as described above),and n=1 to 4; m=1 to 100. The resulting compound-biomolecule conjugatecan have a high binding affinity to a target, for example, due to aninteraction between the biological molecule and the target, for example,via a receptor-ligand interaction, enzyme-substrate interaction, anantibody-antigen interaction, or the like. In other embodiments, suchchemically linked compounds, of the general form [Z¹-(PMB)-Z²]-BM, canbe represented, for example, as:

wherein, in each of the foregoing structures, R₁, R₂, R₃, R₄, R₅, R₆,R₇, R₈, R₉, R₁₀, R₁₃, R₁₄, W₁, W₂, X₁, X₂, and X₃ are as defined herein,Y⁻ is a counterion, and BM is a biomolecule. The foregoing structuresare exemplary and it is understood that a biomolecule (BM) can bechemically linked to such compound via any one or more of the groupsidentified as R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₃, R₁₄, W₁, W₂,X₁, X₂, and X₃

Another aspect of the invention provides a conjugate compound formed byreaction of a biological molecule with a compound a compound describedherein, such as a compound of Formula I-A, I—B, I—C, I-D, or II.

Another aspect of the invention provides a conjugate compound that is acompound described herein (such as a compound of Formula I-A, I-B, I-C,I-D, or II) further substituted with 1, 2, or 3 groups defined by -L-BM;wherein L is a bond or a linker, and -BM is a radical of a biologicalmolecule.

The compounds can be labeled with a biomolecules or cells as follows.The compounds (fluorochromes) of the present invention are incubatedwith one or more biomolecules at various concentrations for about 5minutes to 24 hours or more at a temperature from about 4° C. to about37° C. After the incubation, the free fluorochrome or the fluorochromethat has not been chemically linked to the biomolecule can be removedusing methods known to those skilled in art, such as for example,chromatography or ultrafiltration methods.

Cells can be centrifuged after incubation to create a cell pellet fromwhich the supernatant is removed. Cells can be re-suspended in culturemedia or physiologic saline to wash away residual, unbound or freefluorochrome. This can be repeated several times. In this manner, cellscan be labeled either by direct conjugation to internal or externalcellular molecules or by non-specific cell uptake into variousintracellular compartments, including but not limited to cytosol,endosomes, nucleus, golgi apparatus, and other intracellular organelles.

The disclosed compounds and/or compositions can be packaged as a kit,which may optionally include instructions for using the compounds.Non-limiting examples include kits that contain, for example, acomposition in a powder or lyophilized form, and instructions for using,including reconstituting, dosage information, and storage informationfor in vivo and/or in vitro applications. Kits may optionally containcontainers of a composition in a liquid form ready for use, or requiringfurther mixing with solutions for administration, such as vials forreconstituting powder forms, syringes for injection, customized IVdelivery systems, inhalers, etc. Such containers may contain single ormultiple subject doses. Additionally, a kit can contain components thataid in the detection of the compositions in vivo or in vitro, forexample, specialized endoscopes, light filters.

Compounds disclosed herein, including those compounds chemically linkedto a biomolecule, can be formulated in a pharmaceutical compositionsuitable for administration to a subject, for example, an animal orhuman subject. Accordingly, the formulations include the compoundstogether with a physiologically acceptable carrier suitable for thedesired form and/or dose of administration. Physiologically acceptablecarriers can include water, saline, and may further include agents suchas buffers, and other agents such as preservatives that are compatiblefor use in pharmaceutical formulations. The preferred carrier is afluid, preferably a liquid, more preferably an aqueous solution;however, carriers for solid formulations, topical formulations, inhaledformulations, ophthalmic formulations, and transdermal formulations arealso contemplated as within the scope of the invention.

In addition, the pharmaceutical compositions can include one or morestabilizers in a physiologically acceptable carrier. Suitable example ofstabilizers for use in such compositions include, for example, lowmolecular weight carbohydrates, for example a linear polyalcohol, suchas sorbitol, and glycerol. Other low molecular weight carbohydrates,such as inositol, may also be used.

It is contemplated that the compounds of the invention can beadministered orally or parenterally. For parenteral administration, thecompounds can be administered intravenously, intramuscularly,cutaneously, percutaneously, subcutaneously, rectally, nasally,vaginally, and ocularly. Thus, the composition may be in the form of,e.g., solid tablets, capsules, pills, powders including lyophilizedpowders, colloidal suspensions, microspheres, liposomes granulates,suspensions, emulsions, solutions, gels, including hydrogels, pastes,ointments, creams, plasters, irrigation solutions, drenches, osmoticdelivery devices, suppositories, enemas, injectables, implants, sprays,or aerosols. The pharmaceutical compositions can be formulated accordingto conventional pharmaceutical practice (see, for example, Remington:The Science and Practice of Pharmacy, 20th edition, 2000, ed. A. R.Germaro, Lippincott Williams & Wilkins, Philadelphia, and Encyclopediaof Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, New York).

III Applications of the Fluorochrome Compounds of the Invention

The compounds of the invention can be used in a variety of in vivo andin vitro applications. These applications are discussed in the followingsections.

(a) In Vivo Applications

The invention provides novel fluorescent compounds that can be used in avariety of imaging applications, for example, optical imagingapplications. For a review of optical imaging techniques, see, e.g.,Alfano et al., Ann. NY Acad. Sci. 820:248-270, 1997; Weissleder, NatureBiotechnology 19, 316-317 (2001); Ntziachristos et al., Eur. Radiol.13:195-208 (2003); Graves et al., Curr. Mol. Med. 4:419-430 (2004);Citrin et al., Expert Rev. Anticancer Ther. 4:857-864 (2004);Ntziachristos, Ann. Rev. Biomed. Eng. 8:1-33 (2006); Koo et al., CellOncol. 28:127-139 (2006); and Rao et al., Curr. Opin. Biotechnol.18:17-25 (2007).

An imaging system useful in the practice of this invention typicallyincludes three basic components: (1) an appropriate light source forexciting the fluorochrome compounds of the invention, (2) a system forseparating or distinguishing emissions from light used for inducingfluorochrome excitation, and (3) a detection system. This detectionsystem can be hand-held or incorporated into other useful imagingdevices such as endoscopes, catheters, intraoperative microscopes and/orviewers.

Preferably, the light source provides monochromatic (or substantiallymonochromatic) light. The light source can be a suitably filtered whitelight, i.e., bandpass light from a broadband source. For example, lightfrom a 150-watt halogen lamp can be passed through a suitable bandpassfilter commercially available from Omega Optical (Brattleboro, Vt.).Depending upon the system, the light source can be a laser. See, e.g.,Boas et al., Proc. Natl. Acad. Sci. USA 91:4887-4891, 1994;Ntziachristos et al., Proc. Natl. Acad. Sci. USA 97:2767-2772, 2000; andAlexander, J. Clin. Laser Med. Surg. 9:416-418, 1991. Information onlasers for imaging can be found, for example, at Imaging DiagnosticSystems, Inc., Plantation, Fla. and various other sources. A high passor bandpass filter can be used to separate optical emissions fromexcitation light. A suitable high pass or bandpass filter iscommercially available from Omega Optical, Burlington, Vt.

In general, the light detection system can be viewed as including alight gathering/image forming component and a light detection/imagerecording component. Although the light detection system can be a singleintegrated device that incorporates both components, the lightgathering/image forming component and light detection/image recordingcomponent are discussed separately.

A particularly useful light gathering/image forming component is anendoscope. Endoscopic devices and techniques which have been used for invivo optical imaging of numerous tissues and organs, includingperitoneum (Gahlen et al., J. Photochem. Photobiol. B 52:131-135, 1999),ovarian cancer (Major et al., Gynecol. Oncol. 66:122-132, 1997), colonand rectum (Mycek et al., Gastrointest. Endosc. 48:390-394, 1998; andStepp et al., Endoscopy 30:379-386, 1998), bile ducts (Izuishi et al.,Hepatogastroenterology 46:804-807, 1999), stomach (Abe et al., Endoscopy32:281-286, 2000), bladder (Kriegmair et al., Urol. Int. 63:27-31, 1999;and Riedl et al., J. Endourol. 13:755-759, 1999), lung (Hirsch et al.,Clin Cancer Res 7:5-220, 2001), brain (Ward, J. Laser Appl. 10:224-228,1998), esophagus, and head and neck regions can be employed in thepractice of the present invention.

Other types of light gathering components are catheter-based devices,including fiber optics devices. Such devices are particularly suitablefor intravascular imaging. See, for example, Tearney et al., Science276: 2037-2039, 1997; and Circulation 94: 3013, 1996.

Still other imaging technologies, including phased array technology(Boas et al., Proc. Natl. Acad. Sci. USA 91:4887-4891, 1994; Chance,Ann. NY Acad. Sci. 838:29-45, 1998), optical tomography (Cheng et al.,Optics Express 3:118-123, 1998; and Siegel et al., Optics Express4:287-298, 1999), intravital microscopy (Dellian et al., Br. J. Cancer82:1513-1518, 2000; Monsky et al, Cancer Res. 59:4129-4135, 1999; andFukumura et al., Cell 94:715-725, 1998), confocal imaging (Korlach etal., Proc. Natl. Acad. Sci. USA 96:8461-8466, 1999; Rajadhyaksha et al.,J. Invest. Dermatol. 104:946-952, 1995; and Gonzalez et al., J. Med.30:337-356, 1999) and fluorescence molecular tomography (FMT)(Nziachristos et al., Nature Medicine 8:757-760, 2002; U.S. Pat. No.6,615,063, PCT Application No. WO 03/102558, and PCT US/03/07579) can beused with the fluorochrome compounds of the invention. Similarly, thefluorochrome compounds can be used in a variety of imaging systems, forexample, [1] the IVIS® Imaging Systems: 100 Series, 200 Series (Xenogen,Alameda, Calif.), [2] SPECTRUM and LUMINA (Xenogen, Alameda, Calif.),[3] the SoftScan® or the eXplore Optix™ (GE Healthcare, United Kingdom),[4] Maestro™ and Nuance™-2 Systems (CRi, Woburn, Mass.), [5] ImageStation In-Vivo FX from Carestream Molecular Imaging, Rochester, N.Y.(formerly Kodak Molecular Imaging Systems), [6] OV100, IV100 (OlympusCorporation, Japan), [7] Cellvizio Mauna Kea Technologies, France) [8]NanoSPECT/CT or HiSPECT (Bioscan, Washington, D.C.), [9] CTLM® or LILA™(Imaging Diagnostic Systems, Plantation, Fla.), [10] DYNOT™ (NIRxMedical Technologies, Glen Head, N.Y.) and [11] NightOWL Imaging Systemsby Berthold Technologies, Germany.

A variety of light detection/image recording components, e.g., chargecoupled device (CCD) systems or photographic film, can be used in suchsystems. The choice of light detection/image recording depends onfactors including the type of light gathering/image forming componentbeing used. It is understood, however, that the selection of suitablecomponents, assembling them into an optical imaging system, andoperating the system is within ordinary skill in the art.

Optical imaging and measurement techniques include, but are not limitedto, fluorescence imaging, luminescence imaging; endoscopy; fluorescenceendoscopy; optical coherence tomography; transmittance imaging; timeresolved transmittance imaging; confocal imaging; nonlinear microscopy;photoacoustic imaging; acousto-optical imaging; spectroscopy;reflectance spectroscopy; intravital imaging; two photon imaging;interferometry; coherence interferometry; diffuse optical tomography andfluorescence molecular tomography.

It is contemplated that the fluorochrome compounds of the injection canbe coupled to or incorporated within a solid support, for example, aparticle. Accordingly, it is understood that the fluorochrome compoundscan be coupled to metal oxide nanoparticles that have magneticproperties to produce particles that are also fluorescent. Accordingly,the resulting particles can also be used in MRI imaging using techniquesknown in the art. For a review of MRI techniques see Westbrook, Handbookof MRI Technique, 2^(nd) Edition, 1999, Blackwell Science. It ispossible that images obtained, for example, by fluorescent moleculartomography and by magnetic resonance imaging can be co-registered orfused with one another to provide additional information about the itembeing imaged. Furthermore, multi-modality imaging systems (i.e.,combined optical and MR imaging systems) can be used to create combinedoptical MR images.

In addition, the compositions and methods of the present invention canbe used in combination with other imaging compositions and methods. Forexample, the fluorochrome compounds of the invention can be used toimage regions of interest via optical imaging protocols either alone orin combination with other traditional imaging modalities, such as,X-ray, computed tomography (CT), MR imaging, ultrasound, positronemission tomography (PET), and single photon computerized tomography(SPECT). For instance, the compositions and methods of the presentinvention can be used in combination with CT or MR imaging to obtainboth anatomical and molecular information simultaneously, for example,by co-registration of an image generated by another imaging modality.The compositions and methods of the present invention can also be usedin combination with X-ray, CT, PET, ultrasound, SPECT, MR and otheroptical contrast agents or alternatively, the fluorochrome compounds ofthe present invention may also contain imaging agents, such as iodine,gadolinium atoms and radioactive isotopes, which can be detected usingCT, PET, SPECT, and MR imaging modalities in combination with opticalimaging.

An exemplary method of in vivo optical imaging comprises the steps of(a) administering to a subject, for example, a human or an animal, afluorescent compound of the present invention; (b) allowing sufficienttime for the fluorochrome compound to distribute within the subject orto contact or interact with a biological target; (c) exposing thesubject to electromagnetic radiation, for example, light of a wavelengthabsorbable by the fluorochrome compound; and (d) detecting an opticalsignal emitted by the fluorochrome compound.

It is understood that the subject may be a vertebrate animal, forexample, a mammal, including a human. The animal may also benon-vertebrate, (e.g., C. elegans, drosophila, or other model researchorganisms, etc.). The biological target can include, without limitation,cells, cell culture, tissues, tissue sections, organs, organ sections,cytospin samples, proteins, nucleic acids, carbohydrates, lipids, or thelike.

The foregoing steps, including, for example, steps (a)-(d), can berepeated at predetermined time intervals thereby to permit evaluation ofthe emitted signals of the fluorochrome compounds in the subject overtime. The illuminating and detecting steps (steps (c) and (d),respectively) can be performed using a planar imaging system, endoscope,catheter, tomographic system, hand-held optical imaging system, goggles,or an intraoperative microscope. The signal emitted by the fluorochromecompound can be used to construct an image, for example, a tomographicimage.

Before or during these steps, a detection system can be positionedaround or in the vicinity of a subject (for example, an animal or ahuman) to detect optical and/or other signals (e.g., MR, nuclear, X-ray)emitted from the subject. The emitted optical and/or other signals canbe processed to construct an image, for example, a tomographic or planarimage. In addition, the processed signals can be displayed as imageseither alone or as fused (combined) images.

In addition, it is possible to practice an in vivo imaging method thatselectively detects and images one or more imaging agentssimultaneously. In such an approach, for example, in step (a) notedabove, two or more imaging agents whose signal properties aredistinguishable from one another are administered to the subject, eitherat the same time or sequentially, wherein at least one of the imagingagents contains a fluorochrome compound of the invention. The use ofmultiple agents permits the recording of multiple biological processes,functions or targets.

The invention also features an in vivo imaging method where labeledcells are administered to the subject. The cells can be labeled with thefluorochrome compound ex vivo. The cells can be derived directly from asubject or from another source (e.g., from another subject, cellculture, etc.). The fluorochrome compound can be mixed with the cells toeffectively label the cells and the resulting labeled cells administeredinto a subject in step (a). Steps (b)-(d) then are followed as describedabove. This method can be used for monitoring trafficking andlocalization of certain cell types, including T-cells, tumor cells,immune cells and stem cells, and other cell types. In particular, thismethod may be used to monitor cell-based therapies.

It is understood that the formulation of the fluorochrome compounds, thechoice of mode of administration, the dosages of fluorochrome compoundsadministered to the subject, and the timing between administration ofthe fluorochrome compounds and their exposure of to light (and alsoother forms of electromagnetic radiation if appropriate under thecircumstances) is within the level of skill in the art.

The methods of the invention can be used to determine a number ofindicia, including tracking the localization of the fluorochromecompounds in the subject over time or assessing changes or alterationsin the metabolism and/or excretion of the fluorochrome compounds in thesubject over time. The methods can also be used to follow therapy forsuch diseases by imaging molecular events and biological pathwaysmodulated by such therapy, including but not limited to determiningefficacy, optimal timing, optimal dosing levels (including forindividual patients or test subjects), and synergistic effects ofcombinations of therapy.

The methods and compositions of the invention can also be used to help aphysician or surgeon to identify and characterize areas of disease, suchas arthritis, cancers and specifically colon polyps, or vulnerable orunstable plaque, to distinguish diseased and normal tissue, such asdetecting tumor margins that are difficult to detect using an ordinaryoperating microscope, e.g., in brain surgery, to help dictate atherapeutic or surgical intervention, for example, by determiningwhether a lesion is cancerous and should be removed or non-cancerous andleft alone, or in surgically staging a disease, e.g., intraoperativelymph node staging, sentinel lymph node mapping, or assessingintraoperative bleeding or to delineate tumor margins.

The methods and compositions of the invention can also be used in thedetection, characterization and/or determination of the localization ofa disease, especially early disease, the severity of a disease or adisease-associated condition, the staging of a disease, and/ormonitoring a disease. The presence, absence, or level of an emittedsignal can be indicative of a disease state. The methods andcompositions of the invention can also be used to monitor and/or guidevarious therapeutic interventions, such as surgical procedures, andmonitoring drug therapy, including cell based therapies. The methods ofthe invention can also be used in prognosis of a disease or diseasecondition.

With respect to each of the foregoing, examples of such disease ordisease conditions that can be detected or monitored (before, during orafter therapy) include, for example, inflammation (e.g., inflammationcaused by arthritis, for example, rheumatoid arthritis), cancer (e.g.,colorectal, ovarian, lung, breast, prostate, cervical, testicular, skin,brain, gastrointestinal, pancreatic, liver, kidney, bladder, stomach,leukemia, mouth, esophageal, bone), cardiovascular disease (e.g.,atherosclerosis and inflammatory conditions of blood vessels, ischemia,stroke, thrombosis, disseminated intravascular coagulation),dermatologic disease (e.g., Kaposi's Sarcoma, psoriasis, allergicdermatitis), ophthalmic disease (e.g., macular degeneration, diabeticretinopathy), infectious disease (e.g., bacterial, viral, fungal andparasitic infections, including Acquired Immunodeficiency Syndrome,malaria, Chagas disease, schistosomiasis), immunologic disease (e.g., anautoimmune disorder, lymphoma, multiple sclerosis, rheumatoid arthritis,diabetes mellitus, lupus erythematosis, myasthenia gravis, Gravesdisease), central nervous system disease (e.g., a neurodegenerativedisease, such as Parkinson's disease, Alzheimer's disease, Huntington'sdisease, amyotrophic lateral sclerosis, prion disease), inheriteddiseases, metabolic diseases, environmental diseases (e.g., lead,mercury and radioactive poisoning, skin cancer), bone-related disease(e.g., osteoporosis, primary and metastatic bone tumors,osteoarthritis), neurodegenerative disease, and surgery-relatedcomplications (such as graft rejection, organ rejection, alterations inwound healing, fibrosis, or other complications related to surgicalimplants).

The methods and compositions of the invention, therefore, can be used,for example, to determine the presence and/or localization of tumorcells, the presence and/or localization of inflammation, including thepresence of activated macrophages, for instance in atherosclerosis orarthritis, the presence and in localization of vascular diseaseincluding areas at risk for acute occlusion (i.e., vulnerable plaques)in coronary and peripheral arteries, regions of expanding aneurysms,unstable plaque in carotid arteries, and ischemic areas. The disclosedmethods of the invention can be used, for example, in identification andevaluation of apoptosis, necrosis, hypoxia and angiogenesis.Alternatively, the disclosed methods may also be used to assess theeffect of a therapeutic compound or therapy on a specified moleculartarget by, for example, imaging a subject prior to and after treatmentwith the therapeutic compound or therapy, and comparing correspondingimages.

(b) In Vitro Applications

In addition, it is appreciated that the fluorochrome compounds can alsobe used in a variety of in vitro assays, for example, bindingexperiments, and in vitro imaging experiments. It is understood that theimaging technologies discussed in the previous section are alsoapplicable to in vitro imaging experiments.

An exemplary in vitro imaging method comprises: (a) contacting a samplewith a probe comprising a fluorochrome compound of the invention; (b)allowing the fluorochrome compound to (i) become activated by and/or(ii) bind to a biological target; (c) optionally removing unactivated orunbound fluorochrome compound; (d) exposing the sample toelectromagnetic radiation, for example, light, of a wavelengthabsorbable by the fluorochrome compound; and (e) detecting signalemitted from the fluorochrome compounds thereby to determine whether theprobes have been activated or bound by the biological target.

The sample can be a liquid or solid sample containing, for example,primary cells, cell cultures, or tissue. The biological target can be,for example, a cell, an aggregation of cells, a tissue or tissue sample,a structure (both on the macrocellular level (for example, bone ortissue) or on a subcellular level (for example, a mitochondria ornucleus)), and a cellular component, for example, a protein (forexample, an enzyme or structural protein), lipid, nucleic acid orpolysaccharide.

The fluorochrome compounds can be used in a variety of in vitro ligandbinding assays such, when incorporated into magnetic particles, can beused in magnetic detection based assays (see, U.S. Pat. Nos. 6,046,585and 6,275,031, 5,445,970; 4,219,335, Chemla, et. al. (2000) Proc NatlAcad. Sci USA 97, 14268-72). They can also be used in magnetic resonancebased ligand binding assays such as those described in U.S. Pat. No.5,164,297 and Perez et al. Nature Biotechnol. 2002, 20(8):816-20. Thefluorochrome compounds can also be used for cell sorting and countingapplications.

The fluorochrome compounds can also be used as reporter groups in anucleic acid-based assays. For example, the fluorochrome compounds canbe coupled to nucleic acids, for example, DNA or RNA, modified nucleicacids, PNAs, molecular beacons, or other nucleic acid binding molecules(for example, small interfering RNA or siRNA) for use in hybridizationassays, for example, in situ hybridization assays, sequencing reactions,amplification reactions, for example, real-time polymerase chainreaction amplification reactions. For example, for detecting a singlestranded nucleic acid (i.e., mRNA, cDNA or denatured double-strandedDNA) in a sample via nucleic acid hybridization principles, afluorochrome compound of the invention is chemically linked to asingle-stranded nucleic acid (probe) and contacted with a samplesuspected of containing one or more single stranded nucleic acids(target nucleic acids), optionally immobilized on a solid support. Theprobe is incubated with the sample under conditions to permit the probeto hybridize to target nucleic acid in the sample to form a duplex.Unbound probe can be removed by washing, and the bound probe can bedetected, wherein the presence or level of fluorescence emitted by thefluorochrome compound in the probe is indicative of the presence oramount of the target nucleic acid in the sample.

(c) Ex Vivo Applications

In addition, it is appreciated that the fluorochrome compounds can beused in a variety of ex vivo assays, for example, binding experiments,and ex vivo imaging experiments. It is understood that the imagingtechnologies discussed in the previous sections are also applicable toex vivo imaging experiments.

An exemplary ex vivo imaging method comprises: (a) contacting a samplewith a probe comprising a fluorochrome compound of the invention; (b)allowing the fluorochrome compound to (i) become activated by and/or(ii) bind to a biological target; (c) optionally removing unactivated orunbound fluorochrome compound; (d) exposing the sample toelectromagnetic radiation, for example, light, of a wavelengthabsorbable by the fluorochrome compound; and (e) detecting signalemitted from the fluorochrome compounds thereby to determine whether theprobes have been activated or bound by the biological target.

The sample can be a liquid or solid sample containing, for example,primary cells, cell cultures, or tissue. The biological target can be,for example, a cell, an aggregation of cells, a tissue or tissue sample,a structure (both on the macrocellular level (for example, bone organ ortissue) or on a subcellular level (for example, a mitochondria ornucleus)), and a cellular component, for example, a protein (forexample, an enzyme or structural protein), lipid, nucleic acid orpolysaccharide.

The invention will now be illustrated by means of the followingexamples, which are given for the purpose of illustration only andwithout any intention to limit the scope of the present invention.

EXAMPLES

Representative materials and methods that may be used in preparing thecompounds of the invention are described further below. All commerciallyavailable chemicals and solvents (reagent grade) are used as suppliedwithout further purification in general. Analytical and preparative HPLCmethods include:

A Column: Agilent Zorbax 80 Å, Extend C18, 4.6×250 mm (5 μm).

Mobile phase: Acetonitrile, 25 mM triethylammonium acetate.

B Column: Varian Dynamax, 100 Å, C18, 41.4×250 mm.

Mobile phase: Acetonitrile, 25 mM triethylammonium acetate.

C Column: Phenomenex Jupiter, 300 Å, C18 Mobile phase: Acetonitrile, 25mM triethylammonium acetate.

Example 1—Synthesis of Compound 1g

Synthesis of Compound 1g as the reactive N-hydroxy succinimidyl ester(NHSE) of formula 1 was accomplished through multi step syntheticprocedures as depicted in the scheme 3A below.

Preparation of QS1A:

5-sulfo-2,3,3-trimethyl indolinine as potassium salt (1) was obtainedfrom Syntharo Fine Chemicals, Germany. 10 g of the indolinine (compound1), dried in an oven at 110° C. for a minimum of 3 hrs was reacted with1.5 equivalent of 1,3-propane sultone (TCI America), in 10 mL ofN-methyl pyrrolidinone (Aldrich) by heating in a 100 mL round bottomflask for 8 hrs on an oil bath at 120° C. with constant stirringmagnetically. Yellow reaction mixture turned dark purple and the productprecipitated out of the solution. After cooling to room temp, ethylacetate was added to the reaction mixture (RM) and sonicated for 5 min.The precipitate was filtered, washed three times with ˜100 mL of 90%-10%mixture of ethylacetate (EA)-methanol, and then dried under vacuum for 4hrs. The quaternary salt QS1A obtained in 90% yield was characterized byLCMS (m/e calculated: 361 (as free sulfonic acid); found: 361 (M+1)).

Preparation of Bisanil 9:

Compound 9 was prepared in three steps as shown in the scheme below byfollowing the procedure of Deroover et. al described in the U.S. Pat.No. 5,876,915 (dated Mar. 2, 1999). The intermediates A and B wereisolated by distillation in 13 g and 10 g respectively. Compound B wasconverted to compound 9 by Vilsmeier reaction, and the product wasisolated as dark red solid by filtration and drying under vac for anovernight.

Preparation of Compound 1a:

Compound 9 (100 mg, 0.214 mmol) and compound QS1A (171 mg, 0.418 mmol)were mixed in 2.5 mL acetic acid and 7.5 mL of acetic anhydride. Aftersonicating for two minutes, 35 mg of sodium acetate was added, and themixture was heated at 120° C. with stirring for 4 hrs. Ethyl acetate (25mL) was added, and the solid centrifuged, which was washed with anadditional 5 mL of EA, centrifuged, and the solid dried on speed vac for30 minutes. The crude dye was purified by HPLC on reversed phase (RP)C18 column, using 10-50% triethyl ammonium bicarbonate(TEAB)-acetonitrile (ACN) system. The purified product was characterizedby LCMS. Mass calculated: 968.2 (as free sulfonic acid); Mass found:969.2 (M+1); Yield: 50%.

Preparation of 1b:

To 50 mg of purified compound 1a dissolved in 0.8 mL of distilled waterwas added 0.8 mL of 1M sodium hydroxide, and the reaction mixture wasrotated at room temp in dark. After 90 minutes, 1 mL of 50% aqueousacetic acid was added. Pale yellow reaction mixture turned greenish blueupon acidification. It was purified on RPC18 column, using 10-50%TEAB-ACN system. The pure product was identified to be the mono acidester by LCMS. Mass calculated: 940.2 (as free sulfonic acid); Massfound: 941.1 (M+1); Abs 749 nm; Em 771 nm; ε 240,000 (1×PBS); Yield 80%.

Preparation of 1c:

40 mg of dried compound 1b was dissolved in 0.5 mL of dry DMF in a 2 mLpolypropylene centrifuge tube. 25 mg HATU, 25 mg 2-aminoethanesulfonicacid (Taurine) and 25 uL of N,N-disopropyl ethylamine (DIPEA) were addedand allowed to react at 37° C. for 1 hr. The completion of the reactionwas indicated by LCMS. The crude reaction mixture was diluted with 2 mLof 25% aqueous acetic acid and purified on RPC18 column using 10-40%Triethyl ammonium acetate (TEAAc, pH 6.6)-ACN system. Mass calculated:1047.2 (as free sulfonic acid); Mass found: 1048.1 (M+1). Abs. max: 749nm in water. Yield: 70%.

Preparation of 1d:

30 mg of compound 1c was treated with 250 mM lithium hydroxide solutionat room temp. The saponification was complete in 2 hrs. The resultingacid product was purified on RPC18 column using 5-25% TEAAc-ACN system.Abs. max: 751 nm; Em. Max: 771 nm (in water/1×PBS). Mass calculated:1019.2 (as free sulfonic acid); Mass found: 1020.1 (M+1); Yield: 70%.

Preparation of 1e:

20 mg of dried compound 1d was reacted with a mixture of HATU (20 mg),Ethyl-6-amino hexanoate hydrochloride (25 mg), and DIPEA (15 uL) in DMF(500 uL) at 37° C. for 45 minutes. After diluting with 1 mL of 25%aqueous acetic acid, it was purified by HPLC on RPC18 column using10-40% TEAAc-ACN system. Mass calculated: 1160.3 (as free sulfonicacid); Mass found: 1161.2 (M+1). Abs max: 751 nm; Em. Max: 771 nm (inwater/1×PBS). Yield: 75%.

Preparation of 1f: Compound 1e was treated with 250 mM lithium hydroxidesolution at room temp. The saponification was complete in 1 hr. Theresulting acid product was purified by HPLC on RPC18 column using 5-30%TEAAc-ACN system. Abs. max: 751 nm; Em. Max: 771 nm (in water/1×PBS).Mass calculated: 1132.2 (as free sulfonic acid); Mass found: 1133.3(M+1). Yield: 85%.

Preparation of 1g:

To 5 mg of dried compound 1f was added disuccinimidyl dicarbonate (10mg) and 250 uL dry DMF was added followed by an addition of 5 uLN-methylmorpholine. The NHSE ester formation was complete in about 2 hrsas revealed by a test reaction with butylamine and analyzing byHPLC-LCMS. The NHSE was isolated by precipitation in ethylacetate, andspeed vac drying for 60 min.

The procedure described above for the compounds 1a through 1g are usedfor the compounds synthesized in schemes 3B through 3S.

General Procedure for the Preparation of Quaternary Salts

The N-(propane-3-sulfonate) quaternary salts of indoles, benzindoles,benzoxazoles and benzthiazoles (compounds 2-5, and 10) were prepared byreacting the heterocycles (5 mmol) with 1,3-propane sultone (7.5 mmol)in 1,2-dichlorobenzene or N-methyl pyrrolidonone as indicated in thescheme and heating at 120° C. with stirring for 8 hrs. The productalways formed as solid and was isolated by filtration and washings withsuitable organic solvent mixture (hexane followed by ethylacetate orethylacetate). They were characterized by LCMS.

Similarly the N-Ethyl quaternary salts of the compounds 1-5, and 10 wereprepared by reacting the heterocycles (5 mmol) with ethyliodide (15mmol) in 1,2-dichlorobenzene or N-methyl pyrrolidonone as indicated inthe scheme and heating at 120° C. in a pressure tube for 8 hrs withstirring. The product always formed as solid and was isolated byfiltration and washings with suitable organic solvent mixture. Hexanefollowed by ethylacetate was used for reactions involving 1,2-dichlorobenzene, and only ethylacetate was used for the reactions involvingN-methyl pyrrolidinone. The products were all characterized by LCMS.

The procedure described above for compounds 1a to 1g are followed forthe synthesis of compounds depicted in the synthetic schemes: 3B, 3C,3D, 3E, 3F, 3G, 3H, 3I, and 3J.

Example 2: Synthesis of Asymmetric Dye

Preparation of QS1C:

10 mmol of compound 1 (as acid) was heated with 10 mL of POCl₃ to refluxfor 2 hrs. To the cooled reaction mixture 25 mL n-hexanes were added,and the organic supernatant was safely discarded. The gummy solid wasrotovap dried under vacuum for several hours to remove the residualphosphorous oxychloride. The sulfonlychloride was used as such in thenext step. Yield: 99%.

50 mmol of 4-(N-methyl)-aminobutyric acid hydrochloride was converted toethyl ester by dissolving in 100 mL of absolute ethanol, and carefullyadding thionyl chloride (55 mmol) at room temp with vigorous stirring.The reaction was allowed to proceed over 12 hrs at room temp. Nitrogenwas flushed into the reaction flask and bubbled through the solution for10 min. Solvents were removed by rotovap, and the resulting white solidwas dried under high vacuum for 12 hrs.

The Ethyl (4-(N-methyl))-aminobutyrate hydrochloride as obtained abovewas dissolved in 100 mL dry acetonitrile and cooled to 5° C. 10 foldexcess of triethylamine was added and stirred vigorously. The sulfonylchloride was dissolved in 30 mL of acetonitrile, and was added slowly tothe stirring solution over 10 min during which the solution turnedyellow. Reaction was complete in 30 min. and was allowed to warm up toroom temp. The white triethylamine hydrochloride was filtered off andwashed with cold acetonitrile. The filtrate was concentrated, and theresidue was chromatographed on silica gel using 3% ACN-94% CH₂Cl₂-3% TEAmixture for elution. The product 1C eluted when the eluent used was 5%ACN-92% CH₂Cl₂-3% TEA. It was characterized by LCMS. Yield: 75%.

Compound 1C was converted to the quaternary salt QS1C by following thegeneral procedure described for the synthesis of quaternary salts, using1,2-dichlorobenzene as the solvent. Yield: 75%

General Procedure for the Synthesis of Asymmetric Dyes:

In schemes involving the synthesis of asymmetric dyes using twodifferent quaternary salts derived from two different heterocycles, theprocedure described for compound 1a was followed except that the bisanil(compound 9), the two quaternary slats each were used in equimolaramounts. Everything else remained essentially the same.

Example 3: Conjugation of Compound 1b with BSA

3 mg of BSA (44.4 nmol) was dissolved in 1.5 mL 0.4 M MES buffer at pH5.3, and an aqueous solution of 450 nmoles of compound 1b (45 uL at 10mM) were added followed by 25 mg of EDC. The mixture was left at 37° C.for an overnight (18 hrs). The reaction mixture was diluted with 5 mLwater and filtered through Amicon Ultra-4, PLTK Ultacel-PL Membranefilter with 30 kD cutoff by centrifuging at 2000 rpm for 30 min. Theproduct was washed a few times with 1×PBS buffer until the filtrate wascolorless. The concentrated product was quantified and the dye/proteinratio was determined by the formula:A _(dye)ε_(p)/(A ₂₇₈ −c% A _(dye))ε_(dye)where, A_(dye) is the absorption of the dye at 750 nm, ε_(p) isextinction coefficient of protein (BSA, 43824), A₂₇₈ is the absorptionof the protein at 278 nm, c % A_(dye) is the % absorption of the dye at278 nm with respect to its abs. at λ_(max), 750 nm (4%) and ε_(dye) isthe extinction coefficient of the dye (240,000 in 1×PBS). The productwas also characterized by MALDI (Tuft's University Core Facility,Boston) and the number of dyes was determined to be 8.7 per BSA. Theresults of the fluorescence and absorbance determinations for Compound1b conjugated to BSA are depicted in FIGS. 1A-1B.Schemes

Scheme 1 for the synthesis of quaternary salts, scheme 2 and 2A for thesynthesis of 4,4-disubstituted cyclohexyl bisaldehyde as Schiff's base,and 3B to 3T for the synthesis of dyes of various formulae are shown inthe following pages.

Example 4—Synthesis of Compound 4m (Scheme 3H)

A. Preparation of Compound QS4B

2,3,3-Trimethylbenzindole-5,7-disulfonate (compound 4, 3.1 g, 7 mmol)was dissolved in 25 mL of dry DMF resulting in a clear orange solution.Ethyl iodide, 3 mL (5.85 g, 37.5 mmol, Aldrich) was added and thesolution was heated to 130° C. in a sealed tube for 16 hours. Thereaction mixture, which turned dark purple was cooled and poured into150 mL of ethyl ether. The mixture was centrifuged and the solventdecanted off. The solid product was further washed in the tube withthree 25 mL portions of 2-propanol followed by 25 mL of ether and driedin vacuum. 2.6 g of dark purple solid (85%) was obtained and confirmedby MALDI-TOF-MS. m/e 397.1 [M]+ calculated for C₁₇H₁₉NO₆S₂ ⁺, found397.6.

B. Preparation of Compound 4m

Compound 4m was synthesized using compounds QS4B and 9 through 4h-4l byfollowing the same procedure that was described for the synthesis ofcompound 1f. The overall yield was around 15%. Abs. max: 775 nm (water),780 nm (MeOH); Em. Max: 795 nm (water), 8053 nm (MeOH).

Example 5—Cell Labeling

Mouse splenocytes are prepared as a single cell suspension, and the Tcell subpopulation within the splenocyte preparation are enriched bypassage over a column that removes B cells and macrophages (R&D kit,Mouse T-cell enrichment columns, MTCC500). T cells then are centrifugedto generate a cell pellet of 10⁷ cells. The supernatant is removed fromthe cell pellet, and a solution of 1g at 10 mg/mL (N-hydroxysuccinimideester of Compound 1f) in 100 μL is added. The cells are incubated atroom temperature for 5 minutes, followed by 2 rounds of centrifugationand resuspension in physiological buffer to wash away unbound Compound1f. Cells are assessed by fluorescence microscopy.

Example 6—Cell Labeling and In Vivo Imaging

Mouse 4T1 breast adenocarcinoma cells are centrifuged to generate a cellpellet of 10⁷ cells. The supernatant is removed from the cell pellet,and a solution of 10 mg/mL N-hydroxysuccinimide ester of Compound 1f in100 μL is added. Cells are incubated at room temperature for 5 minutes,followed by 2 rounds of centrifugation and resuspension in physiologicalbuffer to wash away unbound Compound 1f. Cells are assessed byfluorescence microscopy.

Cells are injected intravenously into mice at 5×10⁵ cells per mouse, andlive mice are imaged by fluorescent molecular tomography immediatelyafter injection and 24 hours after injection. As 4T1 cells primarilymetastasize to the lungs, lung fluorescence can be quantified.

Example 7—FMT Imaging with a Compound 1f-Peptide Conjugate

A solution of the N-hydroxysuccinimide ester of Compound 1f ischemically linked to an Arg-Gly-Asp containing peptide under basicconditions to yield a biocompatible fluorescent molecule for in vivooptical imaging.

The tumor cell line HT-29 (human colon carcinoma/HTB-38) is obtainedfrom ATCC (Manassas, Va.). HT-29 cells are grown in McCoy's supplementedwith 10% FBS at 37° C. in a humidified atmosphere containing 5% CO₂.Exponentially growing cells are trypsinized and re-suspended in Hank'sBalanced Salt Solution at a concentration of 3×10⁷ cells/mL. FemaleNU/NU mice 6-8 weeks old (Charles River Laboratory, Wilmington, Mass.)are injected subcutaneously with 3×10⁶ HT-29 cells bilaterally in thefirst mammary fat pads. One week later, when tumors are approximately 30mm³, the mice are injected intravenously with the fluorescent molecule(in 150 μL of 1×PBS) and imaged after 24 hours on a fluorescencereflectance system (FRI, Kodak 2000MM) system and a FluorescenceTomography System (FMT2500) from PerkinElmer, Inc. (Waltham, Mass.).

Example 8—In Vivo Imaging of Bone Growth with Compound 1f

A solution of the N-hydroxysuccinimide ester of Compound 1f ischemically linked to a bisphosphonate containing biomolecule under basicconditions to yield a biocompatible fluorescent molecule for in vivooptical imaging.

Five day-old BALB/c×CF-1 F₁ mice are injected subcutaneously with thefluorescent molecule (in 15 μL 1×PBS) and imaged 24 hours later using afluorescence reflectance imaging (FRI) system (Kodak 2000MM). Areas ofbone growth are imaged.

Example 9—Nanoparticle Labeling

A solution of the N-hydroxysuccinimide ester of Compound 1f ischemically linked to amine groups disposed on a polymeric surface ofiron oxide nanoparticles to yield a biocompatible fluorescent platformfor in vivo fluorescence imaging. Subsequent coupling ofpolyethyleneglycol to these nanoparticles yields a biocompatible imagingagent suitable for fluorescence imaging and intravital microscopy.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications cited herein arehereby expressly incorporated by reference in their entirety and for allpurposes to the same extent as if each was so individually denoted.

EQUIVALENTS

The invention may be embodied in other specific forms without departingform the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A compound of Formula V:

wherein: R₁₁ and R₁₂ are independently: COOH, CONHR, CF₃, halogen, CN,O═C-Phenyl, or COCH₂R; R is H; and Ph is phenyl group, which isoptionally substituted with one of: F, Cl, Br, I, OMe, NMe₂, NO₂, CN,CF₃, or alkyl.
 2. The compound of claim 1, wherein R₁₁ is COOH and R₁₂is COOH.
 3. The compound of claim 1, wherein the compound is:


4. The compound of claim 1, wherein the compound is:


5. The compound of claim 1, wherein the compound is:


6. The compound of claim 1, wherein the compound is the compound ofFormula Va: